Expression Analysis And Subcellular Localization Of The BmNADHb5 Gene From Silkworm, Bombyx Mori

A cDNA sequence containing a conserved domain NDUF-b5 was found from the cDNA library constructed by our laboratory(gene ID: EU826676). We analyzed the bioinformation of a cDNA sequence selected from silkworm pupa cDNA library and found that the cDNA sequence of BmNADHb5 gene contained an ORF of 438 bp encoding a polypeptide of 150 amino acids.In order to prepare the polyclonal antibodies of BmNADHb5, the BmNADHb5 gene was amplified by PCR and inserted into the pET-28a(+) expression vector. Following transformation of E.coli Rosetta cells with the pET-28a(+)-BmNADHb5 construct. Recombinant BmNADHb5 was overexpressed in E.coli Rosetta by adding IPTG for inductor.The recombinant protein was purified with metal-chelating affinity chromatography and then the purified fusion protein was used to immunize a male New Zealand rabbit and generate anti-BmNADHb5 polyclonal antibody. Which titer was larger than 1:12800 when at the concentration of 10μg/mL. We extracted protein and RNA of different stages of Bombyx mori and some tissues of the fifth instar larvae, such as head, epidermis, trachea, Malpighian tubule, fatty body, silk gland, testis, ovary and gut. Then we detected the antibody analyzed by semi-quantitative and Western blot in different stages and tissues. The stage specific expression patterns indicated that BmNADHb5 was expressed the highest level in eggs. The tissue specific expression patterns suggested that BmNADHb5 was expressed the highest level in Malpighian tubule and genital organ. The result of real-time PCR revealed that BmNADHb5 mRNA is widespread in the developmental stages of silkworm, highest in pupa, and different tissues of the fifth instar larva, highest in Malpighian tubule. The subcellular localization of the BmNADHb5 was analyzed. BmNADHb5 accumulated in cytoplasm. All these results will provide us important evidence to make further studies of BmNADHb5.